Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20. We hypothesized that phosphorylation of HSP20 in vascular smooth muscles is associated with alterations in the macromolecular associations of HSP20. Treatment of bovine carotid artery smooth muscles with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and the adenylate cyclase activator, forskolin, led to increases in the phosphorylation of HSP20 and dissociation of macromolecular aggregates of HSP20. However, 3-isobutyl-1-methylxanthine and forskolin treatment of a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle, did not result in increases in the phosphorylation of HSP20 or to dissociation of macromolecular aggregates. HSP20 can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA) in both carotid and umbilical arteries and this phosphorylation of HSP20 is associated with dissociation of macromolecular aggregates of HSP20. Activation of cyclic nucleotide-dependent signaling pathways does not lead to changes in the macromolecular associations of another small heat shock protein, HSP27. Interestingly, the myosin light chains (MLC 20 ) are in similar fractions as the HSP20, and phosphorylation of HSP20 is associated with changes in the macromolecular associations of MLC 20 . These data suggest that increases in the phosphorylation of HSP20 are associated with changes in the macromolecular associations of HSP20. HSP20 may regulate vasorelaxation through a direct interaction with specific contractile regulatory proteins.Agonist pre-contracted bovine carotid artery smooth muscle relaxes with the addition of the guanylyl cyclase activator, sodium nitroprusside, or the adenylyl cyclase activator, forskolin. This relaxation is associated with increases in the phosphorylation of the small heat shock-related protein, HSP20 1 (1). In addition, endothelial-dependent vasodilation of isolated segments of bovine carotid arteries is also associated with increases in the phosphorylation of HSP20 (2). However, HSP20 is not phosphorylated in a muscle that is uniquely refractory to cyclic nucleotide-dependent relaxation, human umbilical artery smooth muscle (3,4). These data suggest that increases in the phosphorylation of HSP20 may mediate cellular signaling processes that lead to vasorelaxation. HSP20 was initially identified as a by-product of the purification of another small heat shock protein, HSP27 (5). HSP27 has been shown to modulate actin filament dynamics in cultured cells (7-9). Increases in the phosphorylation of HSP27 have been associated with vascular smooth muscle contraction (10 -12). HSP20 has been shown to associate in macromolecular aggregates with HSP27, in extracts from heart, diaphragm, and soleus muscle cells (5). Both HSP20 and HSP27 dissociate from the aggregated form in response to heat stress (5, 6). HSP20 and HSP27 are predominant proteins in ...
Vascular diseases, such as atherosclerosis and restenosis following angioplasty or transplantation, are due to abnormal vascular smooth muscle growth and gene expression. The smooth muscle cells (SMC) in response to injury lose their contractile function, become highly proliferative and synthesize and secrete extracellular matrix proteins. Similar changes in the phenotypic properties of vascular SMC occur during in vitro culture. In this report, we examined whether restoration of the expression of the major receptor protein for nitric oxide (NO) signaling in smooth muscle, the guanosine 3′:5′ cyclic monophosphate (cGMP)-dependent protein kinase (PKG), reestablished contractile function to cultured rat aortic SMC. Contractile function was monitored using the silicone polymer wrinkle assay used previously to determine contractility in cultured mesangial cells. Noncontractile rat aortic smooth muscle cells transfected with the cDNA encoding the type I isoform of PKG, but not those transfected with empty vector, formed discreet wrinkles on the substratum in response to serum indicative of contraction. Treatment of the PKG-expressing SMC with sodium nitroprusside (SNP), an NO donor, and with cGMP analogs, or with the adenylyl cyclase activator, forskolin, and with adenosine 3′:5′ cyclic monophosphate (cAMP) analogs reduced wrinkling. The expression of a major PKG substrate protein involved in smooth muscle relaxation, heat shock-related protein-20 (HSP20), was also reestablished in PKG-expressing SMC. Treatment of the PKG-expressing SMC with nitroprusside resulted in phosphorylation of HSP20. Collectively, these results indicate that PKG expression is important to establish contractility to SMC in culture.
Activation of the MAPKAP2 kinase pathway and phosphorylation of HSP27 are associated with thrombin-induced contraction of vascular smooth muscle.
Cyclic nucleotide-dependent vascular relaxation is associated with increases in the phosphorylation of a small heat shock protein (HSP), HSP20. An increase in phosphorylation of another small HSP, HSP27, is associated with impaired cyclic nucleotide-dependent vascular relaxation. Expression of HSPs is altered by exposure to several types of cellular stress in vitro. To determine if behavioral stress in vivo alters vascular expression and phosphorylation of the small HSPs and cyclic nucleotide-dependent vascular relaxation, borderline hypertensive rats were stressed by restraint and exposure to air-jet stress 2 h/day for 10 days or remained in their home cage. Stress impaired relaxation of aorta to forskolin, which activates adenylyl cyclase, and sodium nitroprusside, which activates guanylyl cyclase. This was associated with an increase in the aortic expression and phosphorylation of HSP27, which was localized to the vascular smooth muscle, but a decrease in the amount of phosphorylated (P)-HSP20. To determine if P-HSP27 inhibits phosphorylation of HSP20, P-HSP27 was added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of cAMP-dependent protein kinase. P-HSP27 inhibited phosphorylation of HSP20 in a concentration-dependent manner. These data demonstrate that P-HSP27 can inhibit phosphorylation of HSP20. The increase in P-HSP27 and decrease in P-HSP20 were associated with reduced cyclic nucleotide-dependent vascular smooth muscle relaxation in response to behavioral stress in vivo, an effect similar to that observed previously in response to cellular stress in vitro.
Human umbilical artery smooth muscle is uniquely refractory to cyclic nucleotide-dependent vasorelaxation. Small heat shock proteins (HSPs) have been implicated as contractile regulatory proteins. Thus, we hypothesized that alterations in the phosphorylation of small HSPs may contribute to human umbilical artery smooth muscle vasospasm. Physiologic contractile responses were determined in a muscle bath and compared with phosphorylation events determined with whole-cell phosphorylation and 2-dimensional gel electrophoresis. Precontraction of bovine carotid artery smooth muscle with serotonin followed by relaxation with forskolin was associated with increases in the phosphorylation of HSP27 and HSP20. Precontraction of umbilical artery with serotonin followed by forskolin treatment led to increases in the phosphorylation of HSP27. However, the umbilical artery smooth muscle did not relax, nor was there an increase in the phosphorylation of HSP20 with forskolin treatment. These data suggest that impaired cyclic nucleotide-dependent relaxation of umbilical artery smooth muscle is associated with a lack of phosphorylation of HSP20.
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